U.S. patent number 10,048,266 [Application Number 15/809,555] was granted by the patent office on 2018-08-14 for diagnostic biomarkers and therapeutic targets for pancreatic cancer.
This patent grant is currently assigned to The Johns Hopkins University. The grantee listed for this patent is The Johns Hopkins University. Invention is credited to Robert Anders, Elizabeth A. Jaffee, Darshil T. Jhaveri.
United States Patent |
10,048,266 |
Jaffee , et al. |
August 14, 2018 |
Diagnostic biomarkers and therapeutic targets for pancreatic
cancer
Abstract
We identified >40 proteins that elicited at least a 2-fold
increase in antibody response post-pancreatic-cancer vaccination,
from each of three patients' sera. The antibody responses detected
against these proteins in patients with >3 years disease-free
survival indicates the anti-tumor potential of targeting these
proteins. We found that tissue expression of proteins PSMC5, TFRC
and PPP1R12A increases during tumor development from normal to
pre-malignant to pancreatic tumor. In addition, these proteins were
shown to be pancreatic cancer-associated antigens that are
recognized by post-vaccination antibodies in the sera of patients
that received the vaccine and have demonstrated a favorable disease
free survival.
Inventors: |
Jaffee; Elizabeth A.
(Lutherville, MD), Jhaveri; Darshil T. (Towson, MD),
Anders; Robert (Parkville, MD) |
Applicant: |
Name |
City |
State |
Country |
Type |
The Johns Hopkins University |
Baltimore |
MD |
US |
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Assignee: |
The Johns Hopkins University
(Baltimore, MD)
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Family
ID: |
50883901 |
Appl.
No.: |
15/809,555 |
Filed: |
November 10, 2017 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20180120322 A1 |
May 3, 2018 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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15148674 |
May 6, 2016 |
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14649248 |
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PCT/US2013/072592 |
Dec 2, 2013 |
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61732402 |
Dec 3, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
33/57438 (20130101); G01N 2333/916 (20130101); G01N
2333/705 (20130101); G01N 2333/79 (20130101); G01N
2333/4703 (20130101) |
Current International
Class: |
G01N
33/574 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Gaudernack et al. (Best Practice & Research Clinical
Gastroenterology 2006 vol. 20, p. 299-314) (Year: 2006). cited by
examiner .
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(Year: 2001). cited by examiner .
Berberat et al., Journal of Histochemistry and Cytochemistry.,
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Ryschich, et al., "Transferrin receptor is a marker of malignant
phenotype in human pancreatic cancer and in neuroendocrine
carcinoma of the pancreas", European Journal of Cancer, vol. 40,
No. 9 pp. 1418-1422 (2004). cited by applicant .
Roe et al., "Malignant pleural mesothelioma: genome-wide expression
patterns reflecting general resistance mechanisms and a proposal of
novel targets", Lung Cancer, vol. 67, No. 1 pp. 57-68 (2010). cited
by applicant .
Pan et al., "Protein alterations associated with pancreatic cancer
and chronic pancreatitis found in human plasma using global
quantitative proteomics profiling", Journal of Proteome Research,
vol. 10, No. 5, pp. 2359-2376 (2011). cited by applicant .
Jhaveri et al.,"Abstract 2485: A novel quantitative proteomics
approach to identify proteins that elicit antibody responses in
vaccinated pancreatic cancer patients", Cancer Research, vol. 73,
Issue 8, Supplement 1 (Apr. 15, 2013). cited by applicant .
International Search Report and Written Opinion dated Mar. 14,
2014, for PCT/US2013/072592. cited by applicant.
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Primary Examiner: Cheu; Changhwa J
Attorney, Agent or Firm: Mintz Levin Cohn Ferris Glovsky and
Popeo, P.C. Corless; Peter F.
Government Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with government support under Grant No.
P50CA62924 awarded by the National Institutes of Health/National
Cancer Institute. The government has certain rights in the
invention.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No.
15/148,674, filed May 6, 2016 which is a continuation of U.S.
application Ser. No. 14/649,248, filed on Jun. 3, 2015, which is a
national stage entry of International Application No.:
PCT/US2013/072592, filed on Dec. 2, 2013, which claims priority to
U.S. Provisional Patent Application No. 61/732,402, filed on Dec.
3, 2012, each of which is incorporated herein by reference in its
entirety.
Claims
The invention claimed is:
1. A method of treating a human with a tumor selected from the
group consisting of pancreas, breast, biliary, lung, colon, and
liver, comprising: administering a pancreatic cancer vaccine
composition to the human whereby an immune response to regulatory
subunit 12A of protein phosphatase 1 (PPP1R12A) and/or regulatory
subunit 8 of the 26S proteasome (PSMC5) is raised and measured in
the human.
2. The method of claim 1 wherein prior to said step of
administering a sample of the tumor is tested and expression of
PPP1R12A and/or PSMC5 on cell membranes of the tumor is
detected.
3. The method of claim 1 wherein the vaccine is GM-CSF vaccine
(GVAX).
Description
TECHNICAL FIELD OF THE INVENTION
This invention is related to the area of cancer diagnostics,
prognostics, and therapeutics. Moreover, it relates to the area of
immunotherapeutics.
BACKGROUND OF THE INVENTION
Pancreatic ductal adenocarcinoma is the fourth leading cause of
cancer-related death in the U.S. (1). It is notably the most
aggressive and debilitating malignant disease with a median
survival of less than 6 months. Only 1% to 4% of patients have an
overall survival of more than 5 years (1). Inadequate early
diagnosis, resistance to current therapies, and ineffective
treatment account for these low survival statistics. Alternative
treatment approaches are desparately needed for this disease; the
compelling need for superior treatment options has propelled the
development of new, better-targeted therapies. We have developed an
allogeneic, granulocyte-macrophage colony-stimulating factor
(GM-CSF)-secreting pancreatic cancer vaccine, which has recently
completed phase II clinical trial (2). This promising vaccine is
used in combination with chemoradiation. The observation of
favorable clinical and immunological responses in patients has
testified to the success of the vaccine (2-4). It was shown that
the induction of mesothelin-specific T cell responses only in
patients with a DFS>3 years, which suggests the vaccine induces
immunologically relevant T cell responses (2). Functional genomic
approaches were utilized to identify antigens recognized by T cells
(5). However, finding T cell antigens is limited by the need for
large amounts of patient lymphocytes and the lack of reagents for
each patient-specific HLA (6).
In contrast to T cells, antibodies hold potential as a high
throughput way of identifying antigens. Antibodies can also mount
an effective response against cancer cells through opsonizing,
antigen presentation to T-cells, and mediating cell toxicity via
natural killer cells or the complement system (7). Thus, the
application of seroproteomic approaches has recently gained ground
in the identification of new cancer biomarkers. These cancer
biomarkers are beneficial for both early detection and the
determination of new targets for the development of biologically
relevant therapies (7-12). The most well-known proteomic approaches
utilize sera from untreated cancer patients or individuals with
known genetic susceptibilities for cancer, to screen for
cancer-associated proteins that elicit an antibody response. These
approaches identify oncoproteins that elicit an antibody response
due to differences in expression levels or post-translational
modifications (11). GM-CSF secreting cancer vaccines can also
instigate a broad range of antibody responses, as seen in early
clinical studies (13). Through the study of the immunological
responses in vaccinated patients, we can discover the mechanisms
behind favorable vaccine-induced clinical responses. Identifying
cancer associated proteins will enhance our efforts of identifying
biologically relevant proteins. These proteins have high potential
as future targets for effective pancreatic cancer treatment. This
translational approach will advance the development of new drugs,
vaccines and antibody-based therapies that will halt the
progression and metastasis of the disease. This approach can also
help characterize new proteins that will serve as surrogate
biomarkers, prediction tools of the vaccine's success, and
biomarkers for early diagnosis of pancreatic cancer.
Common proteomic approaches to identify immunogenic proteins are:
Serological Screening of cDNA Expression Library (SEREX),
2-dimensional electrophoresis (2-DE) followed by mass-spectrometry
analysis and protein arrays (7). Proteins found using SEREX and
2-DE approaches are now shown to also elicit T cell responses (6,
13, 14). This provides evidence that antibodies can aid in the
identification of T cell antigens, which further testifies to the
advantages in studying antibodies. SEREX, however, utilizes
proteins expressed in Escherichia coli, which does not account for
human post-translational modifications (12). Contrastingly, the
approach utilizing 2-DE analysis can use human proteins as the
proteome. However, this process has an inherent bias towards
identifying proteins that are abundantly expressed (11). 2D-PAGE
has a lower threshold out of the throughput methods and does not
effectively identify proteins that are very acidic, very basic,
small in size (<15 kDa), or hydrophobic (15). Therefore, this
process is inadequate for detecting membrane-associated proteins,
the most relevant category of proteins as potential biomarkers.
Membrane proteins constitute about 30% of all cellular proteins and
are functionally key regulators (16). In addition, in 2D-PAGE, each
band cut holds several similar molecular weight proteins. This
process is inefficient in separating single proteins, which
obscures which protein instigates the antibody response.
Furthermore, low abundant antigens are generally overshadowed by
high abundant proteins with the same molecular weight in this
process. Both SEREX and SERPA identify linear epitopes, are
relatively low throughput and semi-quantitative (11). Protein
arrays come in many forms. Some protein arrays use tumor cell
lysate fractions, which identify proteins in their native
conformation (11). However, these arrays do not identify which
specific protein in the fraction instigates the immune response and
there also issues with fractionation. The protein arrays with
printed recombinant proteins do not contain human
post-translational modifications because the proteins are expressed
in E. coli or yeast (12). In addition, if a known protein panel is
printed, tumor antigen discovery can be prevented because the
proteome is biased. The protein arrays utilizing printed monoclonal
antibodies are potentially limited by reagent availability thereby
preventing an unbiased proteome being used because a high affinity
and highly specific monoclonal antibody is needed for each protein
to be probed.
There is a continuing need in the art to provide better methods of
early diagnosis, monitoring, prognosing, and treating pancreatic
cancer.
SUMMARY OF THE INVENTION
According to one embodiment of the invention a method detects
pancreatic cancer in a body sample from a human. A body sample is
contacted with at least one antibody that specifically binds to a
protein selected from the group consisting of: Transferrin receptor
(TFRC), regulatory subunit 12A of protein phosphatase 1 (PPP1R12A),
and regulatory subunit 8 of the 26S proteasome (PSMC5). The amount
of antigen bound to the antibody is detected or cellular
localization of the antigen is detected. An increased amount of
antigen bound to the antibody relative to an amount bound to a
control sample or an altered cellular localization indicates the
presence of a pancreatic cancer.
According to another embodiment a method monitors progression of
pancreatic cancer in a body sample from a human. A body sample is
contacted with at least one antibody that specifically binds to a
protein selected from the group consisting of: Transferrin receptor
(TFRC), regulatory subunit 12A of protein phosphatase 1 (PPP1R12A),
and regulatory subunit 8 of the 26S proteasome (PSMC5). The amount
of antigen bound to the antibody is detected. An increased amount
of antigen bound to the antibody relative to an amount bound to a
sample taken at a prior time indicates progression of the
pancreatic cancer. A decreased amount of antigen bound to the
antibody relative to amount bound to a sample taken at a prior time
indicates responsiveness to an anti-cancer treatment.
According to another embodiment a method predicts response to a
pancreatic cancer vaccine in a human. A body sample of the human is
contacted with at least one antibody that specifically binds to a
protein selected from the group consisting of: Transferrin receptor
(TFRC), regulatory subunit 12A of protein phosphatase 1 (PPP1R12A),
and regulatory subunit 8 of the 26S proteasome (PSMC5). The amount
of antigen bound to the antibody is detected. A decreased amount of
antigen bound to the antibody relative to an amount bound to a
control sample prior to vaccination predicts long term disease-free
survival.
According to another embodiment a kit is provided for detecting or
monitoring pancreatic cancer disease or therapy. The kit contains
at least one antibody that specifically binds to an antigen
selected from the group consisting of: Transferrin receptor (TFRC),
regulatory subunit 12A of protein phosphatase 1 (PPP1R12A), and
regulatory subunit 8 of the 26S proteasome (PSMC5). The kit further
contains a detection means for detecting binding complexes of the
antibody and antigens in a test sample.
According to another embodiment a method predicts response to a
pancreatic cancer vaccine in a human. A sample of the human
comprising antibodies is contacted with at least one protein
selected from the group consisting of: Transferrin receptor (TFRC),
regulatory subunit 12A of protein phosphatase 1 (PPP1R12A), and
regulatory subunit 8 of the 26S proteasome (PSMC5). The amount of
antibody bound to the at least one protein is detected. An
increased amount of antibody bound to the at least one protein
relative to an amount bound to a control sample obtained prior to
vaccination predicts long term disease-free survival. According to
another embodiment a kit is provided for detecting or monitoring
pancreatic cancer disease or therapy. The kit comprises at least
one protein selected from the group consisting of: Transferrin
receptor (TFRC), regulatory subunit 12A of protein phosphatase 1
(PPP1R12A), and regulatory subunit 8 of the 26S proteasome (PSMC5).
The kit further comprises a detection means for detecting binding
complexes of the protein with an antibody in a test sample.
According to one embodiment of the invention a method tests a body
sample from a human. A body sample is contacted with at least one
antibody that specifically binds to a protein selected from the
group consisting of: Transferrin receptor (TFRC), regulatory
subunit 12A of protein phosphatase 1 (PPP1R12A), and regulatory
subunit 8 of the 26S proteasome (PSMC5). The amount of antigen
bound to the antibody is detected or cellular localization of the
antigen is detected. An increased amount of antigen bound to the
antibody relative to an amount bound to a control sample or an
altered cellular localization is detected.
According to another embodiment a method tests a body sample of a
human with pancreatic cancer. A first body sample is contacted with
at least one antibody that specifically binds to a protein selected
from the group consisting of: Transferrin receptor (TFRC),
regulatory subunit 12A of protein phosphatase 1 (PPP1R12A), and
regulatory subunit 8 of the 26S proteasome (PSMC5). The amount of
antigen bound to the antibody is detected. The amount detected in
the first body sample is compared to the amount detected in a
second body sample taken from the human at a second time.
According to another embodiment a method tests a body sample of a
human. A first and second body samples of the human are contacted
with at least one antibody that specifically binds to a protein
selected from the group consisting of: Transferrin receptor (TFRC),
regulatory subunit 12A of protein phosphatase 1 (PPP1R12A), and
regulatory subunit 8 of the 26S proteasome (PSMC5). The amount of
antigen bound to the antibody is detected in each sample. The
amount detected in each sample is compared to the other. The first
and second body samples of the human are collected at a first and
second time, wherein the first time is before and the second time
is after the human is vaccinated with a pancreatic cancer vaccine
composition.
According to another embodiment a method tests a body sample of a
human who has received a pancreatic cancer vaccine. A sample of the
human which comprises antibodies is contacted with at least one
protein selected from the group consisting of: Transferrin receptor
(TFRC), regulatory subunit 12A of protein phosphatase 1 (PPP1R12A),
and regulatory subunit 8 of the 26S proteasome (PSMC5). The amount
of antibody bound to the at least one protein is detected. The
amount of antibody detected in the sample of the human who has
received a pancreatic cancer vaccine is compared to the amount
detected in a sample of the human before he or she received the
vaccine.
These and other embodiments which will be apparent to those of
skill in the art upon reading the specification provide the art
with methods and kits for better managing pancreatic cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. The vaccination schedule we used.
FIG. 2. Purification of human antibodies from serum of vaccinated
pancreatic cancer patient. Antibodies (A) were extracted from the
pre-vaccination and post-vaccination sera (S) using a protein G
column.
FIG. 3. Outline of the SASI approach.
FIG. 4. Validation of mass-spectrometry derived SILAC data using
Western-blots. The fold-change detected by mass spectrometry is
shown to the right of each blot.
FIG. 5A-5C. Global changes in antibody recognition post-vaccination
compared to pre-vaccination of patients 9 (FIG. 5A), 27 (FIG. 5B),
and 52 (FIG. 5C), respectively.
FIGS. 6A to 6C. Increased antibody response post-vaccination
correlates with improved survival. Solid arrow shows an increase
post-vaccination whereas a dotted arrow shows a decrease
post-vaccination in antibody response. FIG. 6A: PSMC5; FIG. 6B:
TFRC; FIG. 6C: PPP1R12A
FIG. 7A-7B. PSMC5 staining by immunohistochemistry N: Normal duct
cells, C: Cancer cells FIG. 8A-8C. PPP1R12A staining by IHC. N:
Normal duct cells, C: Cancer cells, I: Isotype control FIG. 9. TFRC
staining by MC. N: Normal duct cells, C: Cancer cells
FIG. 10. Tumor microarrays were scored for the percentage of cells
that express cytoplasmic PPP1R12A (FIG. 10A) or PSMC5 (FIG. 10B).
The distribution of positive staining cells was classified into
<25%, 25%, 50%, 75%, or 100% of tumor cells present. The
frequency of each percentage is plotted in the above histograms.
Tumors with expression patterns significantly different than
pancreas are noted with an * indicated P<0.01.
FIG. 11. Correlation between patient tissue expression of marker
post-surgery and survival post-treatment with vaccine. Higher PSMC5
expression correlates with improved survival post-vaccination FIG.
12. Correlation between patient tissue expression of marker
post-surgery and survival post-treatment with vaccine. Higher
PPP1R12A expression correlates with improved survival
post-vaccination.
DETAILED DESCRIPTION OF THE INVENTION
The inventors have identified three different proteins that are
strongly overexpressed in pancreatic cancer whereas they are either
weakly or not expressed at all in pancreatic normal duct cells.
These proteins are also shown to be targets of a clinically
relevant antibody response induced with a vaccination. Thus, we
have identified candidate proteins as new biomarkers for screening,
and as new targets for therapeutic intervention.
Samples which can be tested include any body sample in which
pancreatic cancer proteins are expressed or shed. These include
without limitation blood, urine, stool, pancreatic tissue samples,
metastatic tissue samples, lymph, lymph nodes.
Any immunological detection technique can be used as is convenient.
These include without limitation ELISA, immunoprecipitation,
immunonblots, radioimmunoassays, protein arrays, and antibody
arrays.
Amounts of antigen can be detected by preparing and comparing to a
standard curve, for example. Amounts may also be determined
relatively, by comparing to a relevant control sample, such as a
sample of the same type obtained from the patient at a different
time, or obtained from a tissue known to be non-cancerous, or a
sample obtained from one or a population of normal patients.
One, two, or three of the identified markers (Transferrin receptor
(TFRC), regulatory subunit 12A of protein phosphatase 1 (PPP1R12A),
and regulatory subunit 8 of the 26S proteasome (PSMC5)) may be used
as a panel. Additional markers including mesothelin, annexin A2,
and galectin 3 may be used. Other clinical parameters may be used
and combined to render a diagnosis or prognosis or assessment of
current or future response to a therapy. The amount of protein
(Transferrin receptor (TFRC), regulatory subunit 12A of protein
phosphatase 1 (PPP1R12A), and regulatory subunit 8 of the 26S
proteasome (PSMC5)) in a sample can be used as a measure of the
disease. Alternatively, the amount of antibody that a patient is
producing to these proteins can be determined as a measure of a
specific and clinically relevant immune response.
Any type of antibody can be used for measurement of protein in a
sample. L Antibodies which can be used to measure proteins may be
polyclonal, monoclonal, single chain, chimeric, or hybrid, for
example. Antibodies can be conjugated to other functionalities to
aid in the detection of the antibodies in an antigen-antibody
complex. Secondary antibodies or radiolabels can be used to detect
antibodies, for example.
Kits can be made with the antibodies or proteins (Transferrin
receptor (TFRC), regulatory subunit 12A of protein phosphatase 1
(PPP1R12A), and regulatory subunit 8 of the 26S proteasome (PSMC5))
useful in carrying out the various described methods. The kits may
have one, two, or three of the described antibodies or proteins.
Additional antibodies or proteins can also be included for further
refinements. Detection means such as enzymes or radiolabels or
secondary antibodies may also be included. Buffers and other
necessary reagents may be included. Instructions may be included in
the kits. The kits' components may be in a divided or undivided
container. A main container may contain sub-containers.
For detection of antibodies in patient samples, preferably the
reagents used will be purified proteins (e.g., Transferrin receptor
(TFRC), regulatory subunit 12A of protein phosphatase 1 (PPP1R12A),
and regulatory subunit 8 of the 26S proteasome (PSMC5)), although
they need not be. The proteins may be made in recombinant cells or
purified from a natural source. The proteins or portions thereof
may be made sythetically.
To overcome the drawbacks of current seroproteomic technologies, we
developed a novel functional proteomic approach that utilizes
high-throughput immunoprecipitation instead of traditional
immunoprecipitation which only utilizes monoclonal antibodies. The
Serum Antibodies based SILAC-Immunoprecipitation (SASI) approach
utilizes immunoprecipitation by serum antibodies, which is then
coupled to quantitative stable isotope labeling by amino acids in
cell culture (SILAC) to identify proteins that elicit a changed
antibody response. Despite the aggressive nature of pancreatic
cancer, seroproteomic approaches have not yet been extensively
applied to studying pancreatic ductal adenocarcinomas (PDA) (9,
10). We utilized a vaccine tumor cell line as the proteome to
analyze immunized sera from pancreatic cancer patients vaccinated
with the GM-CSF vaccine (2). Our study focuses on immunized sera
from patients showing a mesothelin-specific post-vaccination T cell
response correlated with post-vaccination prolonged disease free
survival (2). Using mass spectrometric analysis, the SASI approach
comprehensively identified >45 proteins that elicited at least a
2-fold increase in antibody response post-vaccination. We present
the first large scale study to identify and categorize proteins
that are targeted by antibodies in the human body. The
high-throughput SASI approach identifies both proteins that are of
low abundance as well as in their native state (conformational
epitopes), and provides quantitative measure of the antibody
response, including all changes that would not be apparent by
traditional western blots.
This approach successfully identified a panel of 13 proteins. Three
of these proteins were previously identified by us using the more
crude 2-D gel approach followed by mass spectrometry analysis. This
older approach identified 17 proteins, but only 2 were found to
have biologic importance (Annexin A2 and Galectin-3). As an
example, Annexin A2, was found to be differentially expressed by
pancreatic cancers (6, 18). In addition, we showed that this
protein translocates from the cytosol to the transmembrane through
a tyrosine phosphorylation mechanism that confers metastatic
potential to pancreatic cancer cells (18). Finally, the antibodies
induced by this protein halted metastases. This data provides
evidence that antibody targets have biologic importance to cancer
(6, 18).
The SASI approach was able to identify proteins that were not found
by our prior analysis. Of these proteins, transferrin receptor
(TFRC), regulatory subunit 12A of protein phosphatase 1 (PPP1R12A)
and regulatory subunit 8 of the 26S proteasome (PSMC5) were shown
to be pancreatic cancer associated antigens that are recognized by
antibodies in the sera of vaccinated patients who have demonstrated
favorable disease free survival. We further analyzed PSMC5, TFRC
and PPP1R12A for tissue expression in normal, pre-malignant and
pancreatic tumor specimens and found these proteins increase in
expression with tumor development. Overall, our data demonstrates
that the novel SASI approach can enable identification of candidate
proteins as new biomarkers for screening, prediction tools of the
vaccine's success, and novel targets for therapeutic
intervention.
The above disclosure generally describes the present invention. All
references disclosed herein are expressly incorporated by
reference. A more complete understanding can be obtained by
reference to the following specific examples which are provided
herein for purposes of illustration only, and are not intended to
limit the scope of the invention.
EXAMPLE 1
Materials and Methods
Patients, Serum and Tissue Samples
Patients were enrolled in a phase II study of an allogeneic GM-CSF
secreting whole cell pancreatic cancer vaccine in compliance with
the Johns Hopkins Medical Institution Institutional Review Board
(IRB)-approved J9988 protocol. Blood samples were collected
pre-vaccination, 14 days after 1.sup.st vaccination and 28 days
after each subsequent vaccination. Sera was collected by
centrifugation, aliquoted and stored at -80.degree. C. Pancreatic
tumor tissue samples were obtained from patients prior to
vaccination.
Antibody Purification
Antibodies were purified from pre- and post-3.sup.rd vaccination
sera using a protein G column (GE Healthcare, Piscataway, N.J.,
USA) as per manufacturer's protocol. Quantification of purified
antibodies was done using NanoDrop spectrophotometer (Thermo Fisher
Scientific, Waltham, Mass., USA).
Sample Preparation
The human pancreatic cancer cell line, Panc 10.05 was grown as
previously described. For the SILAC procedure, Panc 10.05 cells
were grown in either light (.sup.12C.sub.6-Lys, .sup.12C.sub.6-Arg)
or heavy (.sup.13C.sub.6-Lys, .sup.13C.sub.6-Arg) RPMI1640 media
containing 10% fetal bovine serum and antibiotics in a humidified
incubator at 37.degree. C. with 5% CO2. Stable isotope containing
amino acids, .sup.13C.sub.6-arginine and .sup.13C.sub.6-lysine,
were purchased from Cambridge Isotope Laboratories (Andover, Mass.,
USA). Arginine and lysine-free RPMI1640 media, fetal bovine serum
(FBS) and antibiotics (penicillin and streptomycin) were purchased
from Invitrogen (Carlsbad, Calif., USA). The light and heavy cells
were washed with phosphate buffered saline and were harvested using
M-PER buffer (Thermo Fisher Scientific) in the presence of cocktail
protease inhibitors (Thermo Fisher Scientific). Protein was
quantified using the Lowry method.
Immunoprecipitation for Mass Spectrometry
Equal amounts of light and heavy cell lysates were incubated
overnight with purified pre- and post-vaccination antibodies,
respectively. On the following day, the two sets of lysate:
antibody mixture were each incubated with protein G beads
(Invitrogen) and washed using M-PER buffer. The immunoprecipitates
were eluted by boiling in NuPAGE.RTM. LDS sample buffer
(Invitrogen). The light and heavy eluted lysates were mixed 1:1.
The mixture was concentrated and resolved by 10% SDS-PAGE. The gel
was stained using a coomassie dye staining kit (Invitrogen).
Liquid Chromatography Tandem Mass Spectrometry and Data
Analysis
The stained gel was excised into 18 bands and each band was
destained in 40 mM ammonium bicarbonate/40% acetonitrile solution.
The samples were reduced with 5 mM dithiothreitol/20% acetonitrile
solution, alkylated with 100 mM iodoacetamide and digested with
trypsin. Sequencing grade modified porcine trypsin was purchased
from Promega (Madison, Wis., USA). The peptides were extracted,
desalted, dried and reconstituted in 0.1% formic acid. The peptides
were analyzed by reversed phase liquid chromatography tandem mass
spectrometry (LC-MS/MS). Briefly, the peptides in solution were
separated using an on-line reverse phase nano high-performance
liquid chromatography using a C18 column and the Eksigent Nano 2D
high-performance liquid chromatography (HPLC) pumping system
(Eksigent). The nano-HPLC is interfaced directly with the
LTQ-Orbitrap-XL (Thermo Electron) allowing for introduction of the
separated peptide solution into the mass spectrometer for tandem
mass spectrometric analysis. Isolated proteins from each band were
identified using an automated database search algorithm, MASCOT,
within the Proteome Discoverer software platform (Thermo Electron)
and processed by MaxQuant. Our data was searched at a mass
tolerance of 10 ppm for MS species and 1 Da for MS/MS with
carbamidomethylation of cysteine as a fixed modification and
oxidation of methionine as a variable modification. The proteolytic
enzyme indicated was trypsin and we allowed up to two missed
cleavage events.
Mass-Spectrometry Data Validation
Panc 10.05 cells grown in light RPMI1640 media were lysed in M-PER
buffer supplemented with protease inhibitor cocktail. The lysate
was immunoprecipitated with either the pre- or post-vaccination
purified antibodies using protein G beads. The immunoprecipitates
were eluted by boiling in NUPAGE LDS sample buffer and resolved on
a NuPAGE 4-12% Bis-Tris gel (Invitrogen). Proteins in the gel were
transferred onto nitrocellulose membrane using a semi-dry apparatus
(Invitrogen). The membrane was blocked in 5% bovine serum albumin
(BSA, Invitrogen) in 0.1% Tween 20-PBS (PBS-T) buffer for 1 hour at
room temperature and probed with the relevant primary antibody
overnight at 4.degree. C. Antibodies against galectin-3 (sc-19283),
E3 ubiquitin protein ligase (sc-9561), mesencephalic
astrocyte-derived neurotrophic factor (sc-34560), epidermal growth
factor receptor kinase substrate 8-like protein 2 (sc-100722),
calpain-1 (sc-81171) were purchased from Santa Cruz Biotechnology
(Santa Cruz, Calif., USA). The membrane was incubated with the
corresponding peroxidase conjugated secondary antibodies (A8419,
Sigma) and then ECL Western Blotting Detection Reagents (GE
Healthcare) was used for 1 minute at room temperature for
developing.
Western Blot for Detecting Antibody Responses in Patients
Purified recombinant proteins, PSMC5 (TP301251), PPP1R12A
(TP323540) and TFRC (TP300980) expressed in human HEK293 cells were
purchased from Origene (Rockville, Md., USA). One microgram of
purified protein was denatured by boiling in SDS-PAGE sample buffer
and resolved on a NuPAGE 4-12% Bis-Tris gel (Invitrogen). Proteins
in the gel were transferred onto nitrocellulose membrane using a
semi-dry apparatus (Invitrogen). The membrane was cut into
individual lanes and was blocked in 5% bovine serum albumin (BSA,
Invitrogen) in 0.1% Tween 20-PBS (PBS-T) buffer for 1 hour at room
temperature. After blocking, each individual lane was probed with
either pre-vaccination or post-vaccination serum of the various
patients at 1:1000 dilution. A lane was used as a control and
probed with mouse anti-DDK antibody (TA150030, Origene) overnight
at 4.degree. C. The membrane was incubated with the peroxidase
conjugated secondary antibodies; goat anti-human IgG antibody
(A8419, Sigma) for patient serum lanes or rabbit anti-mouse IgG
(A9044, Sigma) for control lane. ECL Western Blotting Detection
Reagents (GE Healthcare) was used for 1 minute at room temperature
for developing.
Immunohistochemistry
Immunohistochemistry was performed on formalin-fixed
paraffin-embedded embedded 5 .mu.m thick sections of pancreatic
tumor tissue samples for the available 46 of the 60 patients
enrolled in the study was obtained from the Department of Pathology
at Johns Hopkins Medical Institutions. Standard MC protocol was
applied using Bond-Leica autostainer (Leica Microsystems,
Bannockburn, Ill.). Briefly, tissue sections were baked for 20
minutes at 65.degree. C. followed by deparaffinization, antigen
retrieval and primary antibody incubation at optimal conditions.
Bond polymer detection system was applied to develop the reaction.
3',3' diaminobenzidin (DAB) chromogen-substrate was utilized for
visualization of reaction as per manufacturer's instructions (Leica
Microsystems, Bannockburn, Ill.). All sections were then
counterstained with hematoxylin, dehydrated and cover slipped.
Antibody information is detailed in the table below.
TABLE-US-00001 Name Clone/animal species Dilution Source Anti-PSMC5
Rabbit 1:150 HPA017871, Sigma Anti-PPP1R12A Rabbit 1:500 HPA039443,
Sigma Mouse Mouse (Clone:H68.4) 1:2000 136800, Invitrogen
anti-Human Transferrin Receptor
EXAMPLE 2
Design and Validation of Quantitative Proteomic Approach
60 pancreatic cancer patients, who had their pancreas surgically
removed, were involved in the study (FIG. 1) (2). The patients
received their first vaccination 2 months after surgery. One month
after the first vaccination, the patients underwent a 6-month
course of chemoradiation. The second, third and fourth vaccines
were each administered at sequential one-month intervals from the
time of chemotherapy completion. The fifth, and final, vaccination
was received 6 months after the fourth vaccination. Serum samples
were obtained pre- and post-vaccination for all five vaccinations
(2). The 60 vaccinated patients were divided into 3 groups (A, B
and C) based on length of disease free survival (DFS) (2). Group A
was composed of 12 patients who received all of the scheduled
vaccinations and demonstrated a DFS>3 years (prolonged DFS as
well as overall survival). The clinical time point cutoff was
determined to be 3 years because patients characterized with a
3-year DFS were less likely to have cancer recurrence. The 21
patients in Group B received at least 3 scheduled vaccinations, but
had a DFS<3 years. The 27 patients in Group C relapsed before
receiving their second scheduled vaccination.
EXAMPLE 3
Identification of Proteins by the SASI Approach
To identify the proteins in the post-vaccination sera of patients
in Group A (DFS>3 years), we used the immunized sera from three
patients (patients 9, 27 and 52) who demonstrated other evidence of
post-vaccination immune responses. We identified a total of 976
proteins for patient 9, 811 proteins for patient 27 and 727
proteins for patient 52 (FIG. 5). A broad range of post-vaccination
antibody response was observed; from a 16 fold change increase
post-vaccination to a 10 fold change decrease. The majority of the
proteins, as expected, had no change in response post-vaccination.
We identified 51 proteins for patient 9, 47 proteins for patient 27
and 54 proteins for patient 52 that had a 2 fold change in
response. Through the SASI approach, we present the first large
scale study to identify and categorize proteins that are targeted
by antibodies in the human body.
Pre-vaccination and post-4th vaccination sera from 3 patients,
3.009, 3.027 and 3.052 from Group A was used in the development of
the SASI approach.
The SASI approach consists of 4 key components: (a) Antibody
purification, (b) SILAC labeling, (c) Immunoprecipitation, and (d)
Downstream Analysis.
EXAMPLE 4
(a) Purification of IgGs from Serum
Using a Protein G column, we isolated immunoglobulin G (IgG) from
the serum (FIG. 2). After washing the column, the IgGs are eluted
with a low pH buffer. The eluted IgGs are collected and the pH is
neutralized. Thus, functional pancreatic cancer specific IgGs were
isolated from the immune sera (FIG. 2).
Table 1 shows a partial list of proteins determined to be
biologically relevant in our study. Fold change is defined as the
ratio of post-vaccination to pre-vaccination antibody response.
TABLE-US-00002 Average fold Protein Gene symbol change Protein
function Galectin 3 LGALS3 11.0 Regulator of T-cell functions 26S
proteasome, regulatory PSMC5 4.6 Confers ATP dependency and subunit
8 substrate specificity to the 26S complex MRP-1 CD9 4.1 Cell
adhesion and motility HDGF-2 HDGFRP2 3.2 Function unknown
Centrosomal protein of 170 kDa CEP170 3.1 Microtubule organization
Prohibitin-2 PHB2 2.4 Mediator of transcriptional repression via
recruitment of histone deacetylases Phosphatidylinositol synthase
CDIPT 2.2 Phosphatidylinositol biosynthesis Retinol dehydrogenase
11 RDH11 2.0 Short-chain aldehyde metabolism Aspartate
aminotransferase GOT2 1.9 Amino acid metabolism Protein phosphatase
1, PPP1R12A 1.7 Regulator of protein phosphatase regulatory subunit
12A 1C and mediates binding to myosin Transferrin receptor TFRC 1.7
Iron uptake via endocytosis of transferrin Pyruvate kinase PKM2 1.7
Glycolytic enzyme generating ATP Annexin A2 ANXA2 1.4 Cell
adhesion
Of these proteins, galectin-3, annexin A2 and pyruvate kinase were
identified previously by a 2-D proteomic approach (17). Galectin-3
and annexin A2 are currently under investigation for their role in
pancreatic ductal adenocarcinomas pathogenesis and progression
(18). In our studies to discover biologically relevant proteins in
pancreatic cancer, we have identified the same proteins through two
different proteomic methods. Ongoing research has already shown
these proteins are promising targets involved in signaling pathways
important to the biology of pancreatic cancer progression and
metastasis (17, 18). Therefore, we essentially have ascertained
that our approach determines biologically relevant proteins.
Overall, the SASI approach comprehensively identified more than
2500 proteins.
EXAMPLE 5
(b) SILAC Labeling
The Panc10.05 cell line was utilized in SILAC labeling experiments.
Panc 10.05 is one of the two vaccine tumor cell lines (the
proteome), and its use for SILAC labeling would ensure the antibody
response is specific to human proteins and would contain the
correct post-translational modifications, including glycosylation.
Panc 10.05 was grown in both a heavy version form and a light
version form. Stable isotope labeling with amino acids in cell
culture (SILAC) is a quantitative proteomics method that involves
in vivo labeling of proteins followed by mass spectrometric
analysis. In this method, Panc 10.05 cells incorporate
nonradioactive heavy isotopes of lysines (.sup.13C.sub.6-Lys) and
arginines (.sup.13C.sub.6-Arg) into its proteome instead of the
"light" versions (.sup.12C.sub.6-Lys and .sup.12C.sub.6-Arg)
present in the commercially available media. Panc 10.05 cells were
grown in either "heavy" media containing heavy amino acids or in
"light" media containing normal amino acids. After 9 passages,
cells grown in heavy and light media were lysed to give heavy and
light lysates, respectively.
EXAMPLE 6
(c) Immunoprecipitation
The light and heavy lysates were subjected to overnight
immunoprecipitation, using purified pre- and post-vaccination
antibodies, respectively (FIG. 3). The following day, Protein G
beads were added to capture the IgGs, which were bound to various
proteins from the lysates. Unbound proteins were removed from the
beads by a series of washing steps. Boiling the beads in sample
buffer allowed elution of the immunoprecipitated proteins and IgGs.
This process gave us two sets of samples. One sample consists the
eluted heavy proteins with the post-vaccination IgGs, whereas the
other sample is the eluted light proteins with the pre-vaccination
IgGs. These samples were mixed in a 1:1 ratio. By using equal
amounts of heavy and light protein as well as an equal amount of
antibodies for immunoprecipitation, we are able to infer that the
changes reflected in the heavy to light ratio equates to the
changes in the antibody constitution for each antigen. If a protein
showed increased antibody response post-vaccination, we would see
greater heavy protein to light protein ratio for that protein. If a
protein showed decreased antibody response post-vaccination, we
would see a lower heavy protein to light protein ratio for the
protein.
EXAMPLE 7
(d) Downstream Analysis
The 1:1 heavy and light mixed samples were separated by gel
electrophoresis and stained with coomassie dye. 18 protein bands
were excised and digested with trypsin. The extracted peptides were
analyzed by LTQ-Orbitrap mass spectrometer. The proteins were
identified and quantified using Mascot and MaxQuant,
respectively.
We wanted to further validate the SILAC data derived from
mass-spectrometry analysis. We used pre-vaccination and
post-vaccination antibodies of patient 3.052 for
immunoprecipitation with light cell lysates in both cases (FIG. 4).
Our SILAC data using patient 52 had revealed that galectin-3, E3
ubiquitin-protein ligase UBR5 and mesencephalic astrocyte-derived
neurotrophic factor had an increased antibody response
post-vaccination by 15.3, 4.0 and 3.9 fold respectively.
Contrastingly, this patient also showed a decreased antibody
response post-vaccination for calpain-1 and epidermal growth factor
receptor kinase substrate 8-like protein 2 by 2.5 and 10.0 fold
respectively. To validate our SILAC data, we conducted Western
blots for these proteins. The immunoprecipitated proteins were
separated by SDS-PAGE followed by western blot using antibodies
against the following proteins: galectin-3, E3 ubiquitin-protein
ligase UBR5, mesencephalic astrocyte-derived neurotrophic factor,
calpain-1 and epidermal growth factor receptor kinase substrate
8-like protein 2. We saw that there was a dramatic increase in
galectin-3 protein level in the post-vaccination blot, whereas E3
ubiquitin-protein ligase UBR5 and mesencephalic astrocyte-derived
neurotrophic factor showed a modest increase in detection
post-vaccination. Similarly, calpain-1 showed a dramatic decrease
in detection whereas epidermal growth factor receptor kinase
substrate 8-like protein 2 showed a modest decrease in the blot
containing the post-vaccination immunoprecipitated proteins. The
western blot analysis, though not quantitative, mirrored the trends
we observed from our quantitative mass-spectrometry derived SILAC
ratios.
EXAMPLE 8
PSMC5, PPP1R12A and TFRC are Antibody Targets of Immune Response
Against PDA
Our interest focused on proteins that had greater than 1.5 fold
change response. Previous proteomic approaches had identified
annexin A2 as biologically relevant. In the SASI approach, annexin
A2 revealed a 1.4 fold change in response post vaccination. From
there, we set an average 1.5 fold change post-vaccination with at
least one of the 3 sera tested showing a 2 fold change as our
benchmark for a biologically relevant response. However, some of
these proteins had an increased post-vaccination response in 2 or
all of the sera tested by the SASI approach. We further decided to
test if there was a correlation between the increased
post-vaccination antibody response and disease free status.
Using purified recombinant proteins, we examined the
post-vaccination response in patients with favorable DFS. For this
experiment, we used the serum before the first vaccination as the
pre-vaccination serum, while the serum after the 3.sup.rd
vaccination was designated the post-vaccination serum. PSMC5,
PPP1R12A and TFRC showed elevated antibody titers in patients with
favorable DFS (FIG. 6). PSMC5 elicited an increased antibody
response in 8 of 12 patients. TFRC elicited an increased antibody
response in 8 of 12 patients. PPP1R12A elicited an increased
antibody response in 9 of 12 patients. Interestingly, these 3
proteins also demonstrated an increased antibody response in each
of the 3 patients who were tested in the SASI approach. Although,
we cannot correlate the quantitiative SASI approach data with the
qualitative Western blot results, the overall trends were similar.
This observation further provided validation of our SASI
results.
We then wanted to compare the patients with DFS>3 years to those
with DFS<3 years (FIG. 6). For this comparison, we selected 12
out of the 21 patients in the group with DFS<3 years. The
selection was based on the level of vaccinations completed. Each of
these 12 patients had received at least 3 vaccinations, allowing us
to compare the post-vaccination serum to the pre-vaccination serum.
Western blot analysis showed an increase in antibody response
post-vaccination to recombinant both PSMC5 and TFRC in only 2 of
the 12 patients that showed DFS<3 years (compared to 8 of the 12
patients with DFS>3 years). Western blot analysis showed an
increase in antibody response post-vaccination to recombinant
PPP1R12A in 5 of the 12 patients that showed DFS<3 years
(compared to 9 of the 12 patients with DFS>3 years).
Interestingly, 4 of the 12 patients with DFS<3 years showed a
decreased antibody response to PPP1R12A post-vaccination, whereas
only 1 of the 12 patients with DFS>3 years showed a decreased
response post-vaccination. Similarly, both PSMC5 and TFRC
demonstrated a decreased antibody response in 2 out of the 12
patients DFS<3 years (compared to only 1 patient with DFS>3
years showing a decreased response). These results imply that the
vaccine-induced antibody response to PSMC5, PPP1R12A and TFRC have
strong correlations to clinical benefit. A decreased response
post-vaccination for these proteins is comparable to a shorter DFS.
Data suggests that these proteins are antigenic targets of
vaccine-induced humoral responses in pancreatic cancer patients.
Most significantly, the antibody responses detected against these
proteins in patients with >3 years disease-free survival
suggests an anti-tumor potential of targeting these proteins.
EXAMPLE 9
Increased PSMC5, PPP1R12A, TFRC Tissue Expression Correlates with
PDA Development
Next, we wanted to examine the cause behind the antibody response.
There are 3 main reasons for how these self proteins could induce
an altered antibody response in the patients: difference in
expression levels, difference in localization, or
post-translational modifications. The reports on levels of PSMC5
and PPP1R12A in pancreatic cancer or other cancers are very
preliminary with no extensive information.
First, we analyzed the expression levels and localization of PSMC5
(FIG. 7) in normal as well as cancer tissues by
immunohistochemistry (IHC). The resected tumors for this study came
from 46 of the 60 patients who were treated in our Phase II study
and were available for staining. With immunohistochemistry, we
found that normal pancreatic epithelial ductal cells display weak
cytoplasmic staining for PSMC5. However, ductal carcinoma cells
display strong cytoplasmic staining. PSMC5 is overexpressed in
pancreatic cancer compared to normal tissue. Specifically, 85% of
pancreatic tumor cells have increased expression of PSMC5. PSMC5 is
a part of the 26S proteasome, which is present in all cells;
however, normal cell level of PSMC5 is very low. Normal duct tissue
stained very weakly for PSMC5 (only 15%) in the cytoplasm, with
almost no nuclear staining observed. Additionally, we observed that
PSMC5 localizes to the nucleus in cancer cells, which is shown by
the intense staining in the nucleus of cancer cells. The
cytoplasmic and nuclear expression increases with the progression
from pancreatic intraepithelial neoplasia (PanINs) to PDA. Our data
shows that 50% of the pancreatic tumor cells have increased nuclear
staining of PSMC5. Contrastingly, only 5% of normal duct cells,
acinar cells, blood vessels show nuclear staining of PSMC5. The
isotype controls demonstrated complete negative staining in the 10
slides examined. This data provides evidence that PSMC5 is
overexpressed in PDA and furthermore, the nuclear expression of
PSCM5 increases from normal to cancer tissue. The cancer-specific
increase in PSMC5 provides support to the idea that the protein is
a potential immunologic target.
EXAMPLE 10
Abnormal Subcellular Localization
PPP1R12A or MYPT1 is part of the Rho Kinase pathway component. We
analyzed the expression levels as well as localization of PPP1R12A
(FIG. 8) in normal and cancer tissues by immunohistochemistry (MC).
Through immunohistochemistry, we found that normal pancreatic
epithelial ductal cells display weak cytoplasmic staining.
Contrastingly, the ductal carcinoma cells displayed strong
cytoplasmic staining. PPP1R12A was found to be overexpressed in
pancreatic cancer compared to normal tissue. Specifically, 82% of
the cancer cells have increased expression of PPP1R12A. Only 2% of
normal duct cells stained very weakly for PPP1R12A, with no
membrane staining seen in these cells. We also observed PPP1R12A to
be localized to the membrane and stained strongly and intensely in
the cancer cells. The membrane localization was only observed in
PDA cells. We showed that about 20% of PDA cells have increased
membrane staining of PPP1R12A. On the contrary, the normal duct
cells, acinar cells, blood vessels showed no membrane staining of
PPP1R12A. This data provides support that PPP1R12A is overexpressed
in PDA and that membrane expression of PPP1R12A is a unique feature
of cancer. Thus, PPP1R12A is a potential immunologic target. TFRC
staining were similar to those of PPP1R12A. 74% of PDA cells
stained strongly for TFRC whereas only 1% of the normal duct cells
showed very weak staining (FIG. 9). We also observed some membrane
TFRC staining in only the PDA cells. Similar to PSMC5, both
PPP1R12A and TFRC showed increased staining as the normal duct
cells progressed to the PanIN stages to the full blown PDA
disease.
Thus, the SASI approach has been able to successfully identify
biologically relevant proteins, all 3 of which could be extensively
validated. We saw that each of the 3 markers, PSMC5, PPP1R12A and
TFRC, increases in expression when we compare normal to cancer
cells. Furthermore, there is evidence of mislocalization of these
proteins in cancer. In cancer, PSMC5 is found abnormally in the
nucleus, and PPP1R12A and TFRC are also found on the cell membrane.
Both overexpression and mislocalization in the cancer cells help
explain why an antibody response was targeted towards these
proteins. PSMC5, PPP1R12A, and TFRC have great potential, both as
immunologic targets as well as diagnostic biomarkers. The
heterogeneous nature of both the cancer as well as the antibody
responses illustrates a need for a biomarker panel in order not
only to cover more patients but also retain high specificity.
EXAMPLE 11
Proteins Eliciting Antibody Responses in Vaccinated Pancreatic
Cancer Patients are Expressed by a Range of Adenocarcinomas
Background: Developing targets that identify patients for
appropriate therapies is a key goal of cancer research. A high
throughput proteomic screen identified two proteins, PPP1R12A and
PSMC5, which were found to enhance antibody responses in pancreatic
cancer patients participating in a phase II trial of an allogeneic,
GMCSF-secreting vaccine. Responses to these proteins correlated
with increased disease free survival in trial patients. We sought
to define PPP1R12A and PSMC5 expression in pancreatic and other
common solid malignancies.
Design: Tissue microarrays (TMA) of pancreatic, breast, biliary,
lung, liver, and colon carcinomas were stained for PPP1R12A and
PSCM5. The intensity of tumor cell expression was scored for each
protein from no specific (0); greater than background (1) or strong
(2) staining. The percentage of tumor cells expressing each protein
and the cellular compartment (cytoplasmic, membranous) was
recorded. Positive staining=a score of 1-2 in >25% of cells.
Results: Expression of PPP1R12A was seen in pancreatic (97%),
biliary (58%), colon (46%) ER+ breast (37%) and HER-2+ breast (17%)
adenocarcinomas. Minimal expression was seen in lung (8%) and basal
breast (4%) adenocarcinomas. A higher percentage of pancreatic
cancer expressed PPP1R12A compared to other tumors (p<0.0001).
Significantly more ER+ breast carcinomas expressed PPP1R12A than
HER-2+ or basal type (p<0.001). Membranous PPP1R12A staining was
observed only in pancreas (45%) and colon (30%) cancers. PSMC5
expression was present in all tumors types: pancreatic (57%), ER+
breast (97%), HER-2+ breast (82%), basal breast (86%), liver (69%),
biliary (24%), colon (58%), and lung (74%). Breast tumors showed
particularly high expression of PSMC5. Additionally, HER-2+ tumors
consistently showed expression by 100% of cells within an
individual TMA, which was significantly more than either ER+ or
basal type breast tumors (p<0.01).
Conclusions: Our study confirms strong expression of both PPP1R12A
and PMSC5 in pancreatic cancer. In addition, we identify a range of
adenocarcinomas with expression of PPP1R12A and/or PMSC5 including
breast, biliary, lung, colon, and liver. This identifies tumor
types that might respond to GVAX immunotherapy and provides
rationale to direct therapy based on these proteins expression
patterns. Membranous expression of PPP121RA in pancreatic and colon
cancers is particularly attractive for therapeutic targeting.
Additional studies are needed to evaluate the relationship between
tumor evolution in these adenocarcinomas and the expression of
PPP1R12A and PMSC5.
* * * * *